Toll Like Receptor Ligands Differentially Regulate Expression Of Colonic Epithelial Muscarinic Receptors In a Ligand-Specific and Temporal Manner

NP Hyland, T Inkinen, S Grenham, EK Brint. University College Cork, Cork, Ireland

Introduction: An extraordinary level of cross talk exists between the microbiota and the underlying colonic mucosa, referred to as the microbiota-mucosa interface. Acetylcholine (ACh), a prominent autonomic neurotransmitter, and Toll Like Receptors (TLRs), critical receptors of the innate and adaptive immune system, have been shown to regulate pro-inflammatory cytokine release. Both acetylcholine muscarinic receptors and TLR4 receptors have been implicated in the pathogenesis of inflammatory bowel diseases, though few studies have examined the cross-talk between TLRs and muscarinic receptors. In those studies that have done so, the data largely relate to the airways. Therefore, the aim of this study was to determine whether exposure to TLR3 and TLR4 receptor ligands affects the expression of muscarinic receptors and interleukin 8 (IL-8) in a colonic epithelial cell line, HT29.

Methods: HT29 cells were treated with the TLR3 ligand, PolyI:C (10µM and 25µM) or the TLR4 ligand, lipopolysaccharide LPS (1mg/ml) for either 8 or 24 hrs. qRT-PCR was carried out using probes designed by Applied Biosystems™ to human specific M1, M2 , M3 and IL-8 while using β-actin as an endogenous control. Cycle threshold (Ct) values were recorded. Data were normalised using β-actin and transformed using the 2−ΔCt method. Data are expressed as mean +/− SEM and represent a minimum of three separate experiments. Statistical differences were determined using Student\\'s t-test or One-way ANOVA as appropriate. All tests were performed using GraphPad Prism 5 statistical software.

Results: In HT29 cells, M1 receptor expression increased in a concentration-dependant manner following 8hr preincubation with the TLR3 ligand, PolyI:C (10µM and 25 µM), with the greatest fold-change observed at 8hrs following 25 µM PolyI:C (P < 0.001). Conversely at 8hrs, incubation with the TLR4 ligand, LPS resulted in an approximate 50% decrease in M1 receptor expression (P < 0.05). Both PolyI;C and LPS were shown to induce expression of the chemokine IL-8 at 8hrs (P < 0.0001). The cholinomimetic, bethanechol (BCh; 100µM) significantly reduced M1 receptor expression at 8hrs (P < 0.05). The PolyI:C-induced effect on M1 receptor expression appeared to plateau at 24hrs while LPS stimulation had no effect on M1 receptor expression at this time point. M3 receptor expression, also detectable in HT29 cells, was modestly reduced by PolyI:C at 24hrs, however, neither LPS nor BCh had any effect. M2 receptor expression was undetectable.

Conclusions: These data demonstrate that exposure to viral or bacterial TLR ligands differentially effects M1 receptor expression dependant on the TLR ligand and duration of exposure and is accompanied by a local immune response. Protein expression patterns for the M1 receptor have yet to be confirmed following TLR activation. Nonetheless, the functional effects of microbiota-induced changes in G protein-coupled receptor expression could have both functional and immunological consequences in the colon, particularly in the context of functional and inflammatory bowel disorders.